THE MODEL
It’d be pretty difficult to test the hypothesis in a living human being or other organism. Researchers instead constructed a model to mimic the environment of the blood brain barrier, a model that could be manipulated and observed.
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Making the Model
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A layer of human umbilical vein endothelial cells (HUVECs) were used to mimic endothelial cells in the brain. Taken from umbilical cells shortly after birth, HUVECs are a cheap and readily accessible source of endothelial cells. However, there is one problem with using them, and it lies in the name.
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HUVECs by definition are cells that come from veins. As previously mentioned, the endothelial cells in the blood brain barrier are found in capillaries, which are significantly smaller blood vessels- and therefore significantly more selective when it comes to letting molecules in. Leukocyte travel across these cells wouldn’t be an accurate representation of how they actually travel across the cells in the blood brain barrier.
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One solution to this problem is to artificially make the vein cells more impermeable. Some cells (other than endothelial cells or tight junctions) and molecules support the blood brain barrier and make it more resistant, and might do the same when added to our model. Think of it as giving a warrior defending their kingdom a coat of armor; it's something external we know will make it less likely that an enemy's weapons will harm them.
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These added substances include astrocytes and CPT-cAMP. Both have been shown to increase the activity of tight junctions, making them tighter and less impermeable to outside molecules.
The HUVEC cells of our model were allowed to grow in a solution meant to help cells flourish (a medium) which also contained astrocytes. The goal was to fully incorporate astrocyte cells into the growing HUVEC model so that they could have their intended effect on the cells.
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CPT-cAMP was then added to this ACM/HUVEC mixture to make the model even more impermeable, and the cell model was left undisturbed for 24 h to ensure that all added substances were adequately mixed in. Finally, the cells were placed in plates coated with collagen gel and fibronectin, both common components of cell cultures.
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At this point, the model was a single layer of cells that should have acted much more like the actual blood brain barrier.
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However, it’s not enough to assume that the model acts like the blood brain barrier- it should be tested to prove this is true.
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Testing the Model
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Two assessments were performed to test how impermeable this model was. The more impermeable it was, the more it resembled the actual blood brain barrier, and the more accurate any conclusions made from the experiment would be.
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Electrical Resistance
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Trans endothelial electrical resistance (TEER) was the first.
As the name suggestions, an electrical current was passed through the cell model to determine whether the cells could prevent ions from breaching them. This could be measured by the electrical resistance of the cells- a higher resistance would mean ions would have a harder time getting through the cells.
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The constructed cell model had a resistance of 250 ohms (a common unit of resistance), which was ten times greater than the resistance of the control (shown in the figure to the left) of plain, unaltered HUVEC cells. The resistance was far greater than the known resistance of cells from the actual blood brain barrier (TY10). Since the cell model functions more like the blood brain barrier than actual cells from the BBB (once taken out of their environment), it's a good indication that the model would be best for this experiment.
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Both of these results indicate that the constructed model was as much if not more difficult to breach than the actual blood brain barrier.
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Fluorescence Testing
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The second assessment to measure permeability involved larger molecules: polysaccharides, or complex sugars.
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Fluorescein Isothiocyanate Dextran, a fluorescent polysaccharide, was added to the cell model and left undisturbed for one hour. At the end of the hour, the amount of fluorescence that had permeated the layer of endothelial cells and reached the bottom was measured.
The constructed model (indicated by the red line on the figure to the right) had nearly 10 times less fluorescence at the end of the hour than the control of untouched HUVEC cells, indicating that little had been able to breach the endothelial cells.
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Both assessments indicated that the model was extremely impermeable to ions and other molecules, making it the perfect (or, at least, a good enough) vehicle to test the hypothesis: that more impervious endothelial cells in the blood brain barrier would force leukocytes to cross them in different ways.